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ImmunohistochemistryPrinciples and Applications
Prepared by:Dr/ Ahmed Khalaf AbdelhamidLecturer of Poultry diseases, Faculty of Veterinary medicine, AssiutUniversity
Introduction• Immunohistochemistry (IHC) combines histological,
immunological and biochemical techniques for the
identification of specific tissue components by means of
a specific antigen/antibody reaction tagged with a visible
label.
• IHC makes it possible to visualize the distribution and
localization of specific cellular components within a cell
or tissue.
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Immunohistochemistry utilizes labeled antibodies
to localize specific cell and tissue antigens, and is
among the most sensitive and specific
histochemical techniques.
HISTORY OFIMMUNOHISTOCHEMISTRY
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1945 – Albert Coons 1st used an Ab labeledwith a fluorescent dye to visualize tissues.
1966 – 1st developed enzyme labeling insteadof fluorescent label.
1974 – IHC was performed for the 1st time onroutine formalin fixed paraffin embeddedsections.
1981 – developed avidin-biotin labeling
1991 – Heat induced antigen retrievaltechnique in IHC was done
1995 – Polymer technology introduced
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TERMINOLOGIES
ANTIGENS - Molecules that induces formation ofan Ab and is foreign to the animal into which it isintroduced
Sites on Ag that are capable of inducing Abformation are known as – EPITOPES/ ANTIGENICDETERMINANT – the exact site on the Ag withwhich the Ab combines
•ANTIBODIES – IgG is the most frequently used Ab forIHC•The paratope of Ab binds to the epitope of Ag
•Abs are also proteins - thus any part of the Ab mayitself serve as epitope to induce Ab formation (to whichsecondary Ab binds)
•IHC technique prove that Ig molecules can serve bothas Ab and Ag
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Formalin fixed paraffinembedded sections
Frozen sectionsSmearsImprints
Cytospins
IHC CAN BEPERFORMED ON -
METHODS
• One step staining method• Labeled Ab reacts directly
with Ag in tissueDIRECT
• Unlabeled primary Abreacts with tissue Ag
• Conjugated second Abreacts against primary Ab
INDIRECT
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1. Peroxidase-antiperoxidase method (PAP)2. Biotin-avidin complex method (ABC)3. Labeled streptavidin-biotin method (LSAB)4. Alkaline phosphatase- anti alkaline phosphatase
methos (APAAP)5. Polymer based labeling
Direct Method
It has the advantages of rapidity, ease ofperformance and limited nonspecific reaction.
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Enzymeor fluorescentprimary Ab
antigen
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Indirect Method - Procedure
An unlabeled primary antibody binds tothe tissue antigen.
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primary Ab (mouse)
antigen
Two-Step Indirect Method
An enzyme-labeled secondary antibodybinds to the primary antibody.
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secondary Ab(rabbit anti-mouse)
Enzyme orfluorescent
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Multiple ImmunolabelingMultiple Immunolabeling
IHC PROTOCOL
Fixation and processing
Section cutting
Deparaffinisation and rehydration
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Blocking endogenous peroxidase
Blocking non-specific antibody binding
Antigen retrieval
Primary antibody
Secondary antibody
Chromogen
Chromogen enhancement
Counterstain
Mount
Stringent washingbetween reagents
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Avidin-Biotin Methods• Uses the strong and high affinity of avidin (egg white
glycoprotein) for biotin (water-soluble vitamin).
• Avidin has four binding sites for biotin but fewer thanfour molecules of biotin will actually bind to avidin.
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avidinbiotin
molecules
Avidin-Biotin Methods
• Two of the most common methods include• Avidin-Biotin enzyme Complex (ABC)• Labeled StreptAvidin-Biotin (LSAB)
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• The enzyme complex is prepared by mixingbiotinylated enzyme (HRP or AP) and avidin.
ABC Method
avidin-biotin-enzyme complex
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This preformed avidin-biotin-enzyme complex then reacts with thebiotinylated secondary antibody.
avidinbiotinylatedenzyme
ABC - Procedure
An unlabeled primary antibody bindsto the antigen.
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antigen primary Ab(mouse)
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ABC - Procedure
A biotinylated secondary antibodybinds to the primary antibody.
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secondary Ab(rabbit anti-mouse)
biotin
ABC - Procedure
A preformed avidin-biotin-enzymecomplex solution is added and binds tothe biotinylated secondary antibody.
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avidin-biotin-enzyme complex
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ABC - Procedure
A substrate-chromogen solution isadded ending the reaction andproducing a colored end-product.
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substrate-chromogen
substrate-chromogen
LSAB Method
• Uses enzyme-conjugated streptavidin.Streptavidin is conjugated to severalmolecules of enzyme horseradish peroxidase(HRP) or alkaline phosphatase (AP).
• The secondary antibody is conjugated tonumerous biotin molecules, each of which canpotentially bind to an enzyme-conjugatedstreptavidin.
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LSAB – Procedure
An unlabeled primary antibody bindsto tissue antigen.
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antigen
primary Ab
LSAB – Procedure
A biotinylated secondaryantibody binds to theprimary antibody.
Each secondary antibodycontains multiple biotinmolecules; severalsecondary antibodies canbind to the primaryantibody.
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biotin
secondary Ab
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LSAB – Procedure
An enzyme-labeled streptavidin isadded and binds to the secondaryantibody. HRP-streptavidin
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LSAB – Procedure
A substrate-chromogen solution isadded producing a colored end-product.substrate-
chromogen
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LABELS
ENZYMELABELS
COLLOIDALMETAL LABELS
FLUORESCENTLABELS
RADIOLABELS
ENZYME LABELS
•Mc used labels in IHC are enzymes
•Enzymes used are –
HORSERADISH PEROXIDASE (HRP)
ALKALINE PHOSPHATASE (CALF INTESTINAL)
GLUCOSE OXIDASE
β-D GALACTOSIDASE (BACTERIAL DERIVED)
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CHROMOGENS
AEC and DAB
AEC chromogenMart-1
Melanoma
DAB chromogenMart-1
Melanoma
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Examples of staining results using AEC and DABchromogens.
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Polyclonal Antibodies
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Polyclonal antibodies reacting with various epitopesEach antibody is made by a different B-cell
Monoclonal Antibodies
Monoclonal antibodies reacting with similar epitopes
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APPLICATION OF IHC IN ROUTINESETTINGS
DIAGNOSIS OF TUMORS
PROGNOSTIC MARKER
PREDICTIVE OR THERANOSTIC MARKERS
IDENTIFICATION OF INFECTIOUS ORGANISMS
DIAGNOSIS OFTUMORS
1. Maximum utility of IHC is in distinguishingcarcinoma from lymphoma, sarcoma and melanoma
2. Workup of hematolymphoid neoplasms3. Metastatic carcinoma of unknown primary (CUP)4. Soft tissue neoplasms – 4 common diagnostic setting
a. Small round cell tumorsb. Monomorphic spindle cell tumorsc. Epithelioid soft tissue tumorsd. Pleomorphic spindle cell tumors
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5. In bone – to differentiate primary from metastaticnon –osseous tumors
6. CNS tumors7. Germ cell tumors
1. Loss of myoepithelial or basal cells orbasement membrane/collagen type IV – theseallow assessment of microinvasion
2. Endothelial markers – assist in identification oflymphovascular spaces to ascertain tumorembolism
3. ER, PR and her2/neu4. Ki-67 /MIB-1 – proliferation markers
PROGNOSTICMARKERS
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PREDICTIVE ORTHERANOSTICMARKERS
1. ER/PR – tamoxifen in Ca. breast2. Her 2 – herceptin in breast cancer3. C-kit – gleevac/imatinib in GIST, CML4. CD20 – rituximab in B-cell NHL5. EGFR – erlotinib in lung cancer
IDENTIFICATION OFINFECTIOUSORGANISMS
1. Viruses – HSV, CMV, EBV2. Others – toxoplasma, pneumocystis
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